FVIII expressed by mammalian cells is often specifically or non-specifically absorbed onto cell surfaces by interaction with surface components (e.g. proteoglycans) or by receptor-mediated events (e.g. interaction with LRP receptor). It is also possible that expressed FVIII is enzymatically cleaved and/or degraded in the media of cultured cells. Over time in culture, expressed FVIII concentration decreases in media unless the secreted material is rapidly removed after expression (e.g. by perfusion techniques).
Under ordinary circumstances, the FVIII-vWF complex may be removed from media by conventional chromatographic methods including absorption onto charge matrices or by pseudo-affinity chromatography. FVIII can then be purified away from the FVIII:vWF complex by selective washing steps to yield an enriched population of FVIII molecules, minimally contaminated by vWF.
vWF is formed in the vascular endothelial cells, which are the main source of this plasma protein, by constitutive or stimulated liberation, but it is also synthesized in smaller amounts by the megakaryocytes. It is believed that the primary product of translation is comprised of 2813 amino acids. After cleaving off the signal peptide (22 amino acids), dimerization takes place. Further processing is effected in the Golgi apparatus, the dimers polymerizing after cleavage and removal of the propeptide (741 amino acids). The propeptide plays an important role in the further linking of the dimers, where it catalyses the formation of disulfide bridges at the amino-terminal end. Thus, differently sized oligomers ranging in size from a dimer of 500,000 daltons to large multimers of up to 20 million daltons may form. In addition to the proteolytic procedures, vWF is subject to other post-translational modifications, including glycosylation and sulfation.